Improving protocols for -synuclein seed amplification assays: analysis of preanalytical and analytical variables and identification of candidate parameters for seed quantification

Objectives: The effect of preanalytical and analytical factors on the alpha-synuclein (alpha-syn) seed amplification assay's (SAA) performance has not been fully explored. Similarly, there is limited knowledge about the most suitable assay protocol and kinetic parameters for misfolded alpha-syn seed quantification. Methods: We studied the effect of centrifugation, repeated freeze-thaw cycles (up to seven), delayed freezing, detergent addition, and blood contamination on the performance of the cerebrospinal fluid (CSF) alpha-syn SAA real-time quaking-induced conversion (RT-QuIC). Moreover, we analysed the inter- and intra-plate variability, the recombinant protein batch effect, and the RT-QuIC parameters' variability when multiple samples were run in controlled conditions. Finally, we evaluated the assay potential of quantifying alpha-syn seed by assessing kinetic curves in serial CSF dilutions. Results: Among tested preanalytical variables, a >= 0.01 % blood contamination and adding detergents significantly affected the RT-QuIC kinetic parameters and the number of positive replicates. Increasing the number of replicates improved result reproducibility. The number of positive replicates in serially diluted CSF samples improved discrimination between samples with high and low seeding activity, and the time to threshold (LAG) was the most reliable kinetic parameter in multiple experiment settings. Conclusions: Preanalytical variables affecting alpha-syn RT-QuIC performance are limited to blood contamination and detergent addition. The number of positive replicates and the LAG are the most reliable variables for quantifying alpha-syn seeding activity. Their consistent measurement in serial dilution experiments, especially when associated with an increased number of sample replicates, will help to develop the alpha-syn RT-QuIC assay further into a quantitative test.